In an attempt to clarify the nature of rotavirus antigens and as a means of investigating rotavirus protein structure and function we have isolated a series of monoclones directed at several rotavirus proteins. Monoclones were derived from mice immunized with the Wa strain of human rotavirus (serotype 1), or the rhesus 2 strain of simian rotavirus (RRV) or a human rotavirus (DS-1, serotype 2) reassortant (40-2). We studied 70 monoclones directed at 5 viral proteins. Monoclone production utilized standard techniques and assay systems including RIA, neutralization, immunoprecipitation and hemagglutination-inhibition (HI). The monoclones were characterized by the proteins with which they react: (1) monoclones to the 42,000 dalton inner protein, the 6th gene product; two of these have subgroup specificity. (2) monoclones to the 34,000 dalton surface glycoprotein, the 8th or 9th gene product; some of these neutralize the virus to high titer and some have HI activity. (3) monoclones to the 82,000 dalton surface protein, the 4th gene product, the viral hemmaglutinin; these monoclones inhibit hemagglutination and neutralize virus. (4) monoclones to the 28,000 dalton 10th gene product, the nonstructural glycoprotein; (5) one monoclone that reacts with the 35 KD NS glycoprotein and several monoclones that cannot be classified.